Di(uridine 5&#39;-)tetraphosphate and salts thereof

ABSTRACT

The present invention are directed to P 1 ,P 4 -di(uridine 5′-)tetraphosphate, tetra-alkali metal salts such as tetrasodium, tetralithium, tetrapotassium, and mixed tetra-alkali metal cations thereof. The tetra alkali metal salts of P 1 ,P 4 -di(uridine 5′-)tetraphosphate are water-soluble, nontoxic, and easy to handle during manufacture. These tetra-monovalent alkali metal salts are more resistant to hydrolysis than the mono-, di-, or tri-acid salts, therefore, they provide an improved stability and a longer shelf life for storage. The present invention also provides methods for the synthesis of P 1 ,P 4 -di(uridine 5′-)tetraphosphate, and its pharmaceutically acceptably acceptable salts thereof, and demonstrates the applicability to the production of large quantities. The methods substantially reduce the time required to synthesize diuridine tetraphosphate, for example, to three days or less.

This application is a continuation of U.S. application Ser. No.10/413,965, filed Apr. 14, 2003; which is a continuation-in-part of U.S.application Ser. No. 09/990,546, filed Nov. 20, 2001, U.S. Pat. No.6,548,658, which is a continuation-in-part of U.S. application Ser. No.09/122,516, filed Jul. 24, 1998, U.S. Pat. No. 6,319,908, which claimsthe benefit of U.S. Provisional Application No. 60/054,147 filed Jul.25, 1997. The above applications are incorporated herein by reference intheir entirety.

TECHNICAL FIELD

This invention relates to novel therapeutic dinucleotide saltcompositions and methods for production such. More specifically, itrelates to P¹,P⁴-di(uridine 5′-)tetraphosphate (U₂P₄), tetrasalt and thepharmaceutical formulation thereof.

BACKGROUND OF THE INVENTION

P¹,P⁴-Di(uridine 5′-)tetraphosphate is a dinucleotide of the followingstructure:

The free acid of P¹,P⁴-di(uridine 5′-)tetraphosphate, where X ishydrogen, has been previously described as uridine 5′-(pentahydrogentetraphosphate), P″′→5 ′-ester with uridine (CAS Registry Number:59985-21-6; C. Vallejo et al., Biochimica et Biophysica Acta 438,305(1976) and H. Coste et al., J. Biol. Chem. 262, 12096 (1987)).

Different methods have been described for the synthesis of purinedinucleotides such as diadenosine tetraphosphate (A₂P₄) (E. Rappaport etal, Proc. Natl. Acad. Sci, 78, 838, (1981); A. Guranowski et al,Biochemistry, 27, 2959, (1988); C. Lobaton et al, Eur. J. Biochem., 50,495, 1975; K. Ng and L. Orgel, Nucl. Acid Res., 15, 3573, (1987)).However, this has not been true for U₂P₄ which is a pyrimidinenucleotide. Although purine nucleotides and pyrimidine nucleotidesappear to be analogous, the methods used for purine nucleotide synthesisdo not necessarily work for pyrimidines such as uridine.

Several references describe protocols for the production of diuridinetetraphosphate. The methods described in the literature are very timeconsuming, lasting over five days and producing only small amounts ofdiuridine tetraphosphate (C. Vallejo et al., Biochimica et BiophysicaActa 438, 305 (1976), Sillero et al., Eur J Biochem 76, 332 (1972)).According to this technique, diuridine tetraphosphate was synthesizedthrough a reaction of uridine 5′-monophosphomorpholidate with thetriethylamine salt of pyrophosphoric acid in a medium of anhydrouspyridine. After 5 days at 30° C., pyridine was removed from the reactionmixture by evaporation, and the residue resuspended in glass-distilledwater, the suspension applied to a DEAE-cellulose column andfractionated with a linear gradient (0.06-0.25 M) of ammoniumbicarbonate, pH 8.6. The peak eluting between 0.17-0.19 M ammoniumbicarbonate was partially characterized as U₂P₄ by the followingcriteria: insensitivity to alkaline phosphatase, phosphorus to baseratio and analysis of the products of hydrolysis (UTP+UMP), aftertreatment with phosphodiesterase I, by electrophoresis in citratebuffer, pH 5.0. No yield or spectroscopic data were given. Thus, theseprocedures for the synthesis of diuridine tetraphosphate are lengthy andproduce only small amounts of only partially characterized diuridinetetraphosphate.

Diuridine tetraphosphate has been shown to have beneficial properties inthe treatment of various diseases, such as chronic obstructive pulmonarydisease (COPD). For example, it has been demonstrated to facilitate theclearance of mucous secretions from the lungs of a subject such as amammal including humans in need of treatment for various reasons,including cystic fibrosis, chronic bronchitis, asthma, bronchiectasis,post-operative mucous retention, pneumonia, primary ciliary dyskinesia(M. J. Stutts, III, et al, U.S. Pat. No. 5,635,160; PCT InternationalPublication WO 96/40059) and the prevention and treatment of pneumoniain immobilized patients (K. M. Jacobus and H. J. Leighton, U.S. Pat. No.5,763,447). Further therapeutic uses include treatment of sinusitis(U.S. Pat. Nos. 5,789,391; 5,972,904; 5,981,506; 5,958,897; and PCTInternational Publication WO 98/03177), otitis media (U.S. Pat. No.6,423,694; PCT International Publication WO 97/29756), dry eye (U.S.Pat. No. 5,900,407; PCT International Publication WO 98/34593), retinaldetachment (PCT International Publication WO 02/060454), nasolacrimalduct obstruction (Publication No. US-2002-0103157-A1), the treatment offemale infertility and irritation due to vaginal dryness via increasedmucus secretions and hydration of the epithelial surface (U.S. Pat. No.6,462,028; PCT International Publication WO 00/30629), and enhancing theperformance of athletes.

SUMMARY OF THE INVENTION

The present invention are directed to P¹,P⁴-di(uridine5′-)tetraphosphate, tetra-alkali metal salts such as tetrasodium,tetralithium, tetrapotassium, and mixed tetra-alkali metal cationsthereof. The tetra alkali metal salts of P¹,P⁴-di(uridine5′-)tetraphosphate are water-soluble, nontoxic, and easy to handleduring manufacture. These tetra-monovalent alkali metal salts are moreresistant to hydrolysis than the mono-, di-, or tri-acid salts,therefore, they provide an improved stability and a longer shelf lifefor storage. The present invention also provides methods for thesynthesis of P¹,P⁴-di(uridine 5′-)tetraphosphate, and itspharmaceutically acceptably salts thereof, and demonstrates theapplicability to the production of large quantities. The methods of thepresent invention substantially reduce the time required to synthesizediuridine tetraphosphate, for example, to three days or less.

Another aspect of the present invention is a pharmaceutical formulationcomprising P¹,P⁴-di(uridine 5′)-tetraphosphate, tetra-alkali metal salt,in a pharmaceutically acceptable carrier, in the form of an aqueous, agel, a gel-like, or a solid formulation.

Another aspect of the present invention are methods of treating variousdisease states, including, but not limited to: chronic obstructivepulmonary diseases, sinusitis, otitis media, nasolacrimal ductobstruction, dry eye disease, retinal detachment, pneumonia, and femaleinfertility or irritation caused by vaginal dryness.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for the synthesis of thetherapeutic dinucleotide, P¹,P⁴-di(uridine 5′-)tetraphosphate, and thesalts thereof, which demonstrate large quantity production. The methodsof the present invention substantially reduce the time period requiredto synthesize P¹,P⁴-di(uridine 5′-)tetraphosphate, for example, to threedays or less.

The present invention also provides novel tetra-monovalent alkali metalsalts of P¹, P⁴-di(uridine 5′-)tetraphosphate, irrespective of themethod of making them. The tetra alkali metal salts of P¹,P⁴-di(uridine5′-)tetraphosphate (Formula I) are stable, soluble, nontoxic, and easyto handle during manufacture.

wherein:

each X is independently Li, Na, or K.

Formula I include tetra-monovalent alkali metal salts ofP¹,P⁴-di(uridine 5′-) tetraphosphate, such as tetrasodium, tetralithium,and tetrapotassium salts and tetra-mixed alkali metal salts. Thetetra-mixed alkali metal cations of the tetra-mixed alkali metal saltspreferably are monosodium/tripotassium, disodium/dipotassium,trisodium/monopotassium, monopotassium/trilithium,dipotassium/dilithium, tripotassium/monolithium, monosodium/trilithium,disodium/dilithium, trisodium/monolithium, andmonosodium/monolithium/dipotassium, monosodium/dilithium/monopotassiumand disodium/monolithium/monopotassium cations.

The tetra-alkali metal salts of P¹,P⁴-di(uridine 5′-)tetraphosphate havemany advantages, for example, they provide good long-term stabilityprofiles compared with those of divalent cations (e.g. Ca²⁺, Mg²⁺,Mn²⁺), which catalyze the hydrolysis of phosphate esters. Furthermore,unlike ammonium salts, they cannot release ammonia during long-termstorage, and hence are more stable.

These inorganic sodium, lithium, and potassium cations impart excellentwater solubility compared with hydrophobic amine salts such as tri- andtetrabutylammonium, and similar salts. High water solubility is animportant advantage for flexibility in pharmaceutical formulations ofvarying concentration.

The tetra-alkali metal salts of P¹,P⁴-di(uridine 5′-)tetraphosphate arenon-irritating to the lung and eyes. Other cations may be irritating tothe lungs, eyes, and other mucosal epithelia, or are otherwise not welltolerated by the human body.

These tetra-alkali metal salts of P¹,P⁴-di(uridine 5′-)tetraphosphatehave a high degree (>90%) of purity, thus are suitable forpharmaceutical use. Furthermore, these tetra-alkali metal salts are moreresistant to hydrolysis than the mono-, di-, or tri-acid salts,therefore, they provide an improved stability and a longer shelf lifefor storage. In addition, these salts are easily handled as whitesolids, compared to an oil or gum as with some amine salts.

The present invention provides a pharmaceutical formulation comprisingthe compound of Formula I, in a pharmaceutically acceptable carrier, inthe form of an aqueous, a gel, a gel-like, or a solid formulation. Inone embodiment of the invention, the pharmaceutical formulation is inthe form of an aqueous solution and comprises physiologically safeexcipients formulated to osmolarity between 250-350 mOsm and pH 5-9. Apreferred osmolarity is between about 280 and 320 mOsm, or 280 and 300mOsm. A preferred pH is between about 6.8 and 7.8, or about 7.0 and 7.6.In one embodiment of the invention, the pharmaceutical formulation issterile.

The pharmaceutical formulation comprising tetra-alkali metal salts ofP¹,P⁴-di(uridine 5′-)tetraphosphate (Formula I) can be used tofacilitate the clearance of mucous secretions from the lungs of asubject such as a mammal including humans in need of treatment forvarious reasons, including cystic fibrosis, chronic bronchitis, asthma,bronchiectasis, post-operative mucous retention, pneumonia, primaryciliary dyskinesia (M. J. Stutts, III, et al, U.S. Pat. No. 5,635,160;PCT International Publication WO 96/40059) and the prevention andtreatment of pneumonia in immobilized patients (K. M. Jacobus and H. J.Leighton, U.S. Pat. No. 5,763,447). Further therapeutic uses includetreatment of sinusitis (U.S. Pat. Nos. 5,789,391; 5,972,904; 5,981,506;5,958,897; and PCT International Publication WO 98/03177), otitis media(U.S. Pat. No. 6,423,694; PCT International Publication WO 97/29756),dry eye (U.S. Pat. No. 5,900,407; PCT International Publication WO98/34593), retinal detachment (PCT International Publication WO02/060454), nasolacrimal duct obstruction (Publication No.US-2002-0103157-A1), the treatment of female infertility and irritationdue to vaginal dryness via increased mucus secretions and hydration ofthe epithelial surface (U.S. Pat. No. 6,462,028; PCT InternationalPublication WO 00/30629), and enhancing the performance of athletes.

The compounds of Formula I can be administered orally, topically,parenterally, by inhalation or spray, intra-operatively, rectally, orvaginally in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. The termtopically as used herein includes patches, gels, creams, ointments,suppositiories, pessaries, or nose, ear or eye drops. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques. Inaddition, there is provided a pharmaceutical formulation comprising acompound of general Formula I and a pharmaceutically acceptable carrier.One or more compounds of general Formula I may be present in associationwith one or more non-toxic pharmaceutically acceptable carriers ordiluents or adjuvants and, if desired, other active ingredients. Onesuch carrier would be sugars, where the compounds may be intimatelyincorporated in the matrix through glassification or simply admixed withthe carrier (e.g., lactose, sucrose, trehalose, mannitol) or otheracceptable excipients for lung or airway delivery.

One or more compounds of general Formula I may be administeredseparately or together, or separately or together with: mucolytics suchas DNAse (Pulmozyme®) or acetylcysteine, antibiotics, including but notlimited to inhaled Tobramycin®; non-steroidal anti-inflammatories,antivirals, vaccines, decongestants and corticosteroids.

The pharmaceutical compositions containing compounds of general FormulaI may be in a form suitable for oral use, for example, as tablets,caplets, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsion, hard or soft capsules, or syrups or elixirs.Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients may be, for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example, starch, gelatin oracacia; and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredients is mixed with water oran oil medium, for example, peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example: sodiumcarboxymethylcellulose, methylcellulose and sodium alginate. Dispersingor wetting agents may be a naturally-occurring phosphatide orcondensation products of an allylene oxide with fatty acids, orcondensation products of ethylene oxide with long chain aliphaticalcohols, or condensation products of ethylene oxide with partial estersfrom fatty acids and a hexitol, or condensation products of ethyleneoxide with partial esters derived from fatty acids and hexitolanhydrides. Those skilled in the art will recognize the many specificexcipients and wetting agents encompassed by the general descriptionabove. The aqueous suspensions may also contain one or morepreservatives, for example, ethyl, or n-propyl p-hydroxybenzoate, one ormore coloring agents, one or more flavoring agents, and one or moresweetening agents, such as sucrose or saccharin.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredients inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example, sweetening, flavoring, and coloringagents, may also be present.

Compounds of Formula I may be administered parenterally in a sterilemedium. The drug, depending on the vehicle and concentration used, caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as local anaesthetics, preservatives and buffering agentscan be dissolved in the vehicle. The sterile injectable preparation maybe a sterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent. Among the acceptable vehicles andsolvents that may be employed are sterile water, saline solution, orRinger's solution. The compounds of general Formula I may also beadministered in the form of suppositories for ear, rectal or vaginaladministration of the drug. These compositions can be prepared by mixingthe drug with a suitable non-irritating excipient which is solid atordinary temperatures but liquid at the body temperature and willtherefore melt to release the drug. Such materials are cocoa butter andpolyethylene glycols.

Solutions of compounds of Formula I may be administered byintra-operative installation at any site in the body.

Single dosage levels of the order of from about 1 to about 400 mg,preferably in the range of 10 to 300 mg, and most preferably in therange of 25 to 250 mg, are useful in the treatment of theabove-indicated respiratory conditions. Single dosage levels of theorder of from about 0.0005 to about 5 mg, preferably in the range of0.001 to 3 mg and most preferably in the range of 0.025 to 1 mg, areuseful in the treatment of the above-indicated ophthalmic conditions.The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. It will beunderstood, however, that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, and rate ofexcretion, drug combination and the severity of the particular diseaseundergoing therapy.

The present invention provides the synthesis of diuridine tetraphosphatetetrasodium salt via the tetraammonium salt. In this process, uridine5′-diphosphate disodium salt is first converted to the tributylammoniumsalt using a sulfonic acid resin to generate UDP as the free acid,followed by salt formation with excess tributylamine. After isolation byevaporative water removal, the anhydrous UDP tributylammonium salt isactivated with 1,1-carbonyldiimidazole in DMF solution, during whichtime coupling to form U₂P₄ also occurs. Concentration of the solution invacuo, followed by ion-exchange chromatography on Sephadex DEAE-A25using an increasing gradient of aqueous ammonium bicarbonate yielded asolution of pure diuridine tetraphosphate in ammonium bicarbonatebuffer. The volatile buffer is removed by repeated evaporation withwater on a rotary evaporator to generate the tetraammonium salt of U₂P₄as oil. The oil is dissolved in deionized water to give a ca. 10%solution, and this solution is charged to a Dowex 50Wx4 Na⁺ (Dow) columnand eluted with deionized water. The fractions containing U₂P₄ arepooled, concentrated and lyophilized to yield U₂P₄ tetrasodium salt as awhite solid.

Diuridine tetraphosphate tetrasodium salt has also been synthesized andcrystallized by researchers at Yamasa Corporation using uridinemonophosphate tributylamine salt, diphenylphosphorochloridate andtriethylammonium pyrophosphate (H. Maeda, et al, WO/0020430). The crudematerial is purified by anion exchange chromatography and carbonchromatography to give the diuridine tetraphosphate tetrasodium salt.Crystals of this material are obtained by crystallization from ahydrophilic organic solvent and are suitable for use in x-raycrystallographic analysis of the salt. This reference is incorporatedherein by reference.

The tetrammonium, tetrasodium, tetralithium and tetrapotassium salts ofP¹,P⁴-di(uridine 5′-)tetraphosphate are readily purified by aqueous ionchromatography in which no organic solvents are used.

The monosodium/tripotassium, disodium/dipotassium,trisodium/monopotassium, monopotassium/trilithium,dipotassium/dilithium, tripotassium/monolithium, monosodium/trilithium,disodium/dilithium, trisodium/monolithium,monosodium/monolithium/dipotassium, monosodium/dilithium/monopotassiumand disodium/monolithium/monopotassium salts of P¹,P⁴-di(uridine5′-)tetraphosphate salts of P¹,P⁴-di(uridine 5′-)tetraphosphate areadvantageous in that they are readily obtained in highly purified formdirectly from the di(uridine 5′-)tetraphosphate tetraammonium salt.

An alternative method of making these mixed salts without the use of acation exchange resin is to directly add alkali metal bases to anaqueous solution of the di(uridine 5′-)tetraphosphate tetraammoniumsalt, followed by evaporation of the resulting volatile ammonium saltsunder reduced pressure at about 60° C. or below. This method is analternative method to the current method of preparing tetraammonium,tetrasodium, tetralithium and tetrapotassium salts of P¹,P⁴-di(uridine5′-)tetraphosphate, in that no ion exchange resin is used for theconversion of the tetraammonium salt to the mixed-alkali metal salt, andthus the new process allows for the direct preparation of mixed-alkalimetal salts of di(uridine 5′-)tetraphosphate with specific ratios ofalkali metals present in the composition. The step of using a cationexchange resin is not required in this new procedure.

The synthetic methods described below encompass several syntheticstrategies for producing P¹,P⁴-di(uridine 5′-)tetraphosphate. Generally,all the methods use uridine or uridine nucleotide compounds from FormulaIIa-d as starting materials, which are dissolved in a polar, aproticorganic solvent (e.g. dimethylformamide, dimethylsulfoxide, dioxane,N-methylpyrrolidone, trimethylphosphate) and a hydrophobic amine (e.g.triethylamine, tributylamine, trioctylamine, 2,4,6-collidine,tetrabutylammonium, tri- and tetra-alkyl amines, heterocyclic amines).The product is obtained by phosphorylating with a phosphorylating agentfrom Formula IV (e.g. phosphorus oxychloride, pyrophosphate,pyrophosphorylchloride) or activating a phosphate group with anactivating agent from Formula III (e.g. carbonyldiimidazole, an alky oraryl carbodiimide, an alkyl or aryl phosphochloridate), respectively,with subsequent purification various means well known to those of skillin the art, including, but not limited to, ion chromatography (e.g. DEAESephadex, DEAE cellulose, Dowex 50, anion and cation exchange resins).Each tetra-alkali metal salt is generated from the purifiedtetraammonium salt using alkali metal bases that generate ammonia orvolatile salts in the presence of ammonium ions (e.g. sodium hydroxide,sodium bicarbonate, sodium acetate, potassium carbonate, potassiumbicarbonate, potassium acetate, lithium formate) in a ratio whichprovides the desired tetra-alkali metal salt of P¹,P⁴-di(uridine5′-)tetraphosphate after evaporation of the volatile salts and materialspresent.

The method of synthesizing compounds of Formula I is carried outgenerally by the following steps: 1) dissolving uridine or uridinenucleotide compounds of Formulas IIa-d in a polar, aprotic organicsolvent and a hydrophobic amine; 2) phosphorylating with aphosphorylating agent of one of the Formulas IVa-b to yield an aminesalt of a compound of Formula IA, or activating a phosphate group of theuridine nucleotide compound with an activating agent of one of theFormulas IIIa-c and reacting with a suitable compound of Formula II b-dto yield an amine salt of a compound of Formula IA; 3) purifying by ionexchange chromatography using a volatile ammonium-salt buffer such asammonium bicarbonate, ammonium formate or ammonium acetate; and 4)evaporating the volatile aqueous buffer solution and re-evaporating withwater several times to give the pure tetraammonium salt. An aqueoussolution of this ammonium salt is treated with between zero and fourequivalents of sodium ion, in the form of sodium hydroxide, -alkoxide,-formate, -acetate, -trifluoroacetate, -carbonate or -bicarbonate base,and between zero and four equivalents of potassium ion, in the form ofpotassium hydroxide, -alkoxide, -formate, -acetate, -trifluoroacetate,-carbonate or -bicarbonate base, and between zero and four equivalentsof lithium ion, in the form of lithium hydroxide, -alkoxide, -formate,-acetate, -trifluoroacetate, -carbonate or -bicarbonate base, such thata total of four equivalents of alkali metals are present for eachequivalent of diuridine tetraphosphate and the four equivalents ofammonium ion present in diuridine tetraphosphate tetraammonium salt. Theaqueous solution of the desired mixed salt of U₂P₄ shown in Formula isthen evaporated to dryness to remove volatile ammonium salts and solventunder vacuum. Water is added and the material is concentrated at below60° C. on a rotary evaporator under vacuum several times to removetraces of ammonia, volatile ammonium salts and solvent. Water is addedto give an approximately 10% aqueous solution, and this is lyophilizedto give the desired tetra salt of diuridine tetraphosphate.

The pyrimidine β-D-ribofuranosyl starting materials uridine, uridine5′-monophosphate (UMP), uridine 5′-diphosphate (UDP), and uridine5′-triphosphate (UTP) are shown as free acids in Formulas IIa-d below,respectively. These materials are all commercially available in largequantity in various salt forms.

and salts thereof,

and salts thereof;

and salts thereof.

The activating agents carbodiimide, activated carbonyl, and activatedphosphorus compounds are shown in the general Formulas IIIa-c below,respectively.

wherein R₁ and R₂ are C₁-C₈ alkyl or cycloalkyl, C₁-C₈ optionallysubstituted alkyl or cycloalkyl (e.g. hydroxy and amino groups); aryl oroptionally substituted aryl (e.g. hydroxy and amino groups). Preferredcompounds of Formula IIIa are dicyclohexylcarbodiimide and1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

wherein X is imidazole, tetrazole, and/or halogen. Preferred compoundsof Formula IIIb are carbonyldiimidazole and carbonylditriazole.

wherein R₁ and R₂ are C₁-C₈ alkyl or cycloalkyl, C₁-C₈ optionallysubstituted alkyl, alkoxy or cycloalkyl (e.g. hydroxy and amino groups);aryl, alkoxy or optionally substituted aryl or alkoxy (e.g. hydroxy andamino groups) and/or halogen; and X is halogen. Preferred compounds ofFormula IIIc are diphenylphosphorochloridate, phenylphosphorodichloridate, phenylphosphonic dichloride anddiphenylphosphinic chloride.

The mono- and diphosphorylating agents are shown below in the generalformulas IVa-b.

wherein X is halogen. Preferred compound of Formula IVa is phosphorusoxychloride.

wherein X is oxygen, hydroxy, or halogen, and salts thereof. Preferredcompounds of Formula IVb are pyrophosphoryl chloride and pyrophosphate.

Those having skill in the art will recognize that the present inventionis not limited to the following examples and that the steps in thefollowing examples may be varied.

EXAMPLES Example 1 Method for the Production of DiuridineTetraphosphate, Tetrasodium Salt using Uridine 5′-Diphosphate

Uridine 5′-diphosphate disodium salt (Yamasa, Choshi, Japan; 600 grams)was dissolved in deionized water (5.4 L). The solution was passedthrough a Dowex 50Wx4 H⁺ (Dow Chemical) column. The fractions containinguridine 5′-diphosphate were pooled and neutralized with tributylamine(Aldrich, St. Louis; 300 mL). The neutralized fractions wereconcentrated to an oil by using a rotary evaporator at a bathtemperature of 55-60° C. The oil was dissolved in dry dimethylformamide(Aldrich, 3 L) and then dried by concentrating to an oil using a rotaryevaporator (55-60° C. bath temperature). This step was repeated twice.The oil was again dissolved in dimethylformamide (3 L) and1,1-carbonyldiimidazole (Aldrich; 100 g) was added. The solution washeated at 50° C. for 2½ hours. An additional amount of activating agent(33 grams) was added and heating continued for a further 2½ hours. Thesolution was again concentrated to an oil on a rotary evaporator (bathtemperature at 55-60° C.). The resulting oil was dissolved in deionizedwater to a conductivity equal to that of 0.2 M NH₄HCO₃. The solution wasthen loaded into a column of Sephadex DEAE-A25 (Pharmacia, Upsala,Sweden; pre-swollen in 1.0 M NaHCO₃ and washed with 2 column volumes ofdeionized H₂O). The column was eluted with the following solutions inthe following order: 60 L of 0.25 M NH₄HCO₃, 120 L of 0.275M NH₄HCO₃, 40L of 030 M NH₄HCO₃ and 40 L of 0.35 M NH₄HCO₃. The fractions havingsufficient amounts of pure diuridine tetraphosphate were pooled asdetermined by HPLC analysis and concentrated on a rotary evaporator(bath temperature at 55-60° C.). The resulting residue was dissolved indeionized water (1.5 L) and concentrated on a rotary evaporator. Thisstep was repeated 15 times or until excess of bicarbonate buffer wasremoved. The resulting oil was dissolved in a sufficient amount ofdeionized water to form a ca. 10% solution, the solution charged to aDowex 50Wx4 Na⁺ (Dow) column and eluted with deionized water. Thefractions containing U₂P₄ were pooled and concentrated to a ca. 10-15%solution, which was lyophilized to yield U₂P₄ tetrasodium salt as awhite solid (150 g approximately 25% yield based on uridine5′-diphosphate).

Structure Elucidation of P¹,P⁴-di(uridine 5′-)tetraphosphate,Tetrasodium Salt

Due to the lack of adequate spectroscopic data of nonadenylateddinucleotides in the literature, a full structure elucidation ofP¹,P⁴-di(uridine 5′-)tetraphosphate, tetrasodium salt was performed byemploying modern analytical techniques. The molecular weight wasdetermined by mass spectrometry to be 878 [m/z 855, (M—Na⁺)⁻],confirming the molecular formula C₁₈H₂₂N₄O₂₃P₄.4Na. The exact massmeasured for C₁₈H₂₂N₄O₂₃P₄.3Na [(M—Na⁺)⁻: calculated 854.9318] was854.9268. The measured mass differed from the theoretical mass by 5.0milimass units (5.9 ppm) for a confidence level of 99.7%. Karl Fishermoisture analysis gave a value of 1.73% H₂0 and further confirmation ofthe molecular formula was obtained from elemental analysis: calculatedfor Na=10.70, found 10.81%; C:P ratio calculated 1.74, found 1.80, basedon the molecular formula: C₁₈H₂₂N₄O₂₃P₄.4.2Na.1.1H₂O (FW=902.4 g/mol).The infrared spectrum showed a broad signal at 3422 cm⁻¹ and a signal at1702 cm⁻¹, indicating the presence of hydroxyl (O—H stretch) andcarbonyl (C═O stretch) functional groups. In addition, a phosphate P═Ostretch was observed at 1265 cm⁻¹. The UV spectrum in water displayed aλ_(max) of 262 nm with a molar absorptivity of 17,004. The specificrotation at 25° C. (c=1, H₂O) was determined by polarimetry to be−9.56°.

The NMR spectra are: ¹H NMR (D₂O, TMS) δ 4.11 (m, 2H), 4.14 (m, 1H),4.25 (m, 1H), 4.27 (m, 1H), 5.84 (d, J=8.1 Hz, 1H), 5.86 (d, J=5.4 Hz,1H), 7.81 (d, J=8.1 Hz); ¹³C NMR (D₂O, TMS) δ 65.1 (d, J=5.5 Hz), 69.7,73.5, 83.4 (d, J=9.4 Hz), 88.1, 102.8, 141.5, 152.9, 167.5; ³¹P NMR(D₂O, H₃PO₄ std) δ −22.32 (m), −10.75 (m). The ¹H coupled ³¹P spectrumshowed a broadening of the multiplet at δ −10.75 ppm due to theintroduction of ¹H coupling. This multiplet was therefore confirmed asP_(α). There was no effect of ¹H coupling on the multiplet at −22.23ppm, assigning this by default as P_(β). A Nuclear Overhauser Effect(NOE) was observed for H₆ to the H_(2′) and H_(3′) sugar protons.Because it is not possible for H₅ to show an NOE to the sugar protons,H₆ is confirmed. Additionally, N₁ substitution is confirmed, because nopyrimidine-sugar NOE is possible for an N₃ substituted structure.

Additional 2-dimensional NMR experiments were conducted to verifyconnectivity. HMQC shows connectivity for H₅ to C₅ and H₆ to C₆,confirming C₅ and C₆. COSY and NOE connectivity were observed for H₅ toH₆, verifying H₅. HMBC 3-bond connectivity was observed for: H₆ toC_(1′), C₆ to H_(1′), H_(1′) to C₂, H₆ to C₂. These data thus confirmH₁, C₂ and N₁ substitution. COSY connectivity of H_(1′) to H_(2′)confirms H_(2′) and HMQC connectivity of H_(1′) to C_(1′) and H_(2′) toC_(2′) confirms C_(1′) and C_(2′). Additionally, HMBC shows 2-bond Jconnectivity from H₅ to C₄, confirming C₄. A ¹³C DEPT spectrum withmult=1.5 shows the carbon at δ 65.1 inverted relative to all othercarbons. This observation confirms that C_(5′) is a methylene. Thecoupling of ³¹P to carbons at δ 65.1 and 83.4 confirms C_(5′) andC_(4′), because C_(4′) is the only coupled methyne. In addition, HMQCshows connectivity for C_(5′) to H_(5′) and C_(4′) to H_(4′), confirmingH_(4′) and H_(5′). An NOE was observed for H_(1′) to H_(4′), H₆ toH_(2′) and H₆ to H_(3′), confirming the β anomer sugar configuration.

In conclusion, P¹,P⁴-di(uridine 5′-)tetraphosphate, tetrasodium salt wassynthesized on a 150 g scale in 25% yield from commercially availablestarting materials with a total reaction time of 5 hours. The crudeproduct was efficiently purified by ion exchange chromatography and thestructure of the reaction product was unambiguously proven using massspectroscopic, NMR and other analytical techniques.

Example 2 Method for the Production of Diuridine TetraphosphateTetrammonium Salt using Uridine 5′-Monophosphate

Uridine 5′-monophosphate (Sigma, Milwaukee, 3.0 g, 9.26 mmol) wasdissolved in dry DMF (10 mL) and tributylamine (Aldrich, 2 mL). Thesolution was evaporated in vacuo at 40° C. to an oil. The residue wasdissolved in dry DMF (Aldrich, 8 mL) to form a solution.Carbonyldiimidazole (Aldrich, 1.65 g, 10.18 mmol) was added to thissolution. The reaction was heated at 50° C. for one hour. Uridine5′-triphosphate (Yamasa, 5.60 g, 10.18 mmol) prepared as the anhydroustributylammonium salt in DMF (5 mL) and tributylamine (2 mL), asdescribed in Example 3 below, was added to the reaction solution. Themixture was allowed to stir at 50° C. for three days when the solutionwas evaporated in vacuo to an oil, redissolved in water (5 mL) andpurified by column (300×50 mm) chromatography (Sephadex DEAE-A25,40-120μ, Aldrich, pre-swollen in 1.0 M NaHCO₃ and washed with 2 columnvolumes of deionized H₂O (H₂O→0.3 M NH₄HCO₃ gradient). The purefractions were concentrated in vacuo at 35° C., and H₂O added andreevaporated 5 times to obtain diuridine tetraphosphate tetrammoniumsalt as a white solid (2.37 g,30% yield): 92.11% pure by HPLC with thesame retention time as the standard. In addition, the tetrammonium saltwas analyzed by FABMS to give a mass of [C₁₈H₂₅N₄O₂₃P₄ (M—H⁺)⁻:calculated 788.9860] 788.9857, confirming a parent formula ofC₁₈H₂₆N₄O₂₃P₄ for the free acid].

Example 3A Method for the Production of Diuridine Tetraphosphate usingUridine 5′-Triphosphate (UTP)

A solution of uridine 5′-triphosphate (UTP) trisodium salt (ProBioSint,Varese, Italy; 5.86 g, 0.01 mol) in water (5 mL) was passed through acolumn of BioRad AG-MP 50 (Aldrich) strong cation exchange resin in itstributylamine form (50 mL bed volume) and eluted with distilled water(about 300 mL). To this solution was added tributylamine (Aldrich; 5mL), and the suspension shaken until the pH of the aqueous fraction hadrisen to 8. The layers were separated and the aqueous solutionevaporated to small volume, then lyophilized overnight. The residue wasdissolved in dry dimethylformamide (Aldrich; 20 mL) and the solventevaporated at 0.1 mmHg. The dried tributylamine salt was made up to 100mL with anhydrous acetone to yield a stock solution (0.1 M in UTP).Dicyclohexylcarbodiimide (DCC) (Baker, Phillipsburg; 0.227 g, 1.2 mmol)was added to an aliquot of the foregoing UTP solution (10 mL, 1.0 mmol)and the solution stirred at room temperature for 30 minutes. The mixturewas added to the triethylamine salt of uridine 5′-monophosphate (2.0mmol, prepared by addition of triethylamine (0.5 mL) to a solution ofuridine 5′-monophosphate (UMP) (Sigma; 0.648 g in DMF), and evaporatingto dryness). This suspension was then evaporated to dryness, the residuemade up to 5.0 mL in dry DMF, and set aside at 40° C. for 24 hours. Thereaction mixture was separated by semipreparative ion-exchangechromatography (Hamilton PRP X-100 column), eluting with a gradient of0-1.0 M ammonium bicarbonate, 5 mL/min, 30 minutes. The dinucleotidetetraphosphate eluted between 21 and 23 minutes; the product (76.7%yield based on UTP) was quantitated by comparison of its ultravioletabsorption at λ_(max) 263 nm with that of a standard solution ofP¹,P⁴-di(uridine 5′-)tetraphosphate.

Example 3B Method for the Production of Diuridine Tetraphosphate usingUridine 5′-Triphosphate (UTP) and an Excess of Activating Agent

Conversion of UTP to P¹,P⁴-di(uridine 5′-)tetraphosphate can be enhancedby activation of the tributylamine salt (0.1 mmol) with a large excessof DCC (0.1 g, 0.5 mmol); in this case the deposited dicyclohexylureawas removed by filtration, the reaction mixture extracted with ether (10mL) and the residue dissolved in dry DMF prior to treatment withtributylamine UMP (0.2 mmol). Upon chromatographic separation of thereaction mixture and quantitation by ultraviolet absorption as inExample 3A above, the uridine tetraphosphate product constituted 50.7%of the uridylate species in the mixture, corresponding to a conversionfrom UTP of 95.9%.

Example 4A Method for the Production of Diuridine Tetraphosphate usingUridine 5′-Monophosphate Activated with Carbonyldiimidazole

Uridine 5′-monophosphate (UMP) (0.324g, 1.0 mmol) was dissolved in amixture of dry DMF (5 mL) and tributylamine (237 μL, 1 mmol) thesolution was evaporated to dryness, then twice more with DMF to yieldthe anhydrous tributylamine salt. The residue was dissolved in DMF (5mL) and carbonyldiimidazole (CDI) (0.81 g, 5 mmol) added. The solutionwas set aside for 3 hours, then methanol 324 μL, 8 mmol) added todestroy the excess of CDI. The solution was set aside for one hour.Tributylamine pyrophosphate (Sigma, 0.228 g, 0.5 mmol) was added and thesuspension stirred under nitrogen at room temperature. After 3 hours thereaction was quenched with water and the mixture subjected to HPLC as inExample 3A above. Yield of P¹,P⁴-di(uridine 5′-)tetraphosphate asquantitated by its absorbance at 263 nm was 9.3%.

Example 4B Method for the Production of Diuridine Tetraphosphate usingUridine 5′-Monophosphate Activated with Diphenyl Phosphochloridate

The anhydrous tributylamine salt of UMP (1.0 mmol), prepared essentiallyas above, was dissolved in a mixture of dry dioxane (5 mL) and DMF (1mL). Diphenyl phosphochloridate (0.3 mL) and tributylamine (0.3 mL) wereadded and the solution set aside at room temperature for 3 hours. Thesolvent was evaporated and the residue shaken with ether (˜10 mL), thenset aside at 4° C. for 30 minutes. The ether was decanted and theresidue was dissolved in a solution of tributylamine pyrophosphate(0.228 g, 0.5 mmol) in DMF (3 mL). The solution was stored undernitrogen at room temperature. After 3 hours the reaction was quenchedwith water and the mixture subjected to HPLC as in Example 3A above.Yield of P¹,P⁴-di(uridine 5′-)tetraphosphate as quantified by itsabsorbance at 263 nm was 9.6%.

Example 5 Method for the Production of Diuridine Tetraphosphate usingUridine, Phosphorus Oxychloride and Pyrophosphate

Uridine (Aldrich, 0.244 g, 1 mmol) was dissolved in trimethyl phosphate(Aldrich, 5 mL) and tributylamine (466 uL, 2 mmol) added. The solutionwas stirred at 0 degrees during the addition of phosphorus oxychloride(0.153 g (93.2 uL), 1 mmol), and the resulting suspension stirred at 0°C. for 3 hours. Tributylamine pyrophosphate (0.228 g) was added and thesuspension stirred at room temperature for 3 hours. The reaction wasquenched with 1.0 M aqueous triethylamine bicarbonate and the mixtureextracted with methylene chloride to remove trimethyl phosphate. Theaqueous solution was subjected to HPLC as in Example 3A above.Conversion of uridine to P¹,P⁴-di(uridine 5′-)tetraphosphate asquantitated by absorbance of the latter at 263 nm was 6.83%.

Example 6 Method for the Production of Diuridine Tetraphosphate usingUridine 5′-Monophosphate and Pyrophosphoryl Chloride

Uridine 5′-monophosphate (UMP) (64.8 mg, 0.2 mmol) was dissolved in drypyridine (1 mL) and stirred in ice during the addition of pyrophosphorylchloride (13.9 uL (25 mg), 0.1 mmol). The solution became cloudy almostimmediately, then a copious semicrystalline white precipitate formedwhich became a gummy mass within 1-2 minutes. The mixture was stored atroom temperature overnight, the quenched with water and subjected toHPLC as in Example 3A above. Yield of P¹,P⁴-di(uridine5′-)tetraphosphate as quantitated by its absorbance at 263 nm was 15.8%.A substantial amount of P¹,P³-di(uridine 5′-)triphosphate (25.4%) wasobtained as the major by-product.

Example 7 Aqueous Stability and Solubility of P¹,P⁴-di(uridine5′-)tetraphosphate, Tetrasodium Salt

The solubility of P¹,P⁴-di(uridine 5′-)tetraphosphate, tetrasodium saltin water was determined by adding portions of solid to a known volume ofdeionized water until the solution became turbid. The maximum solubilityin water was thus determined to be ca. 900 mg/mL. Stability studies ofthe solid or aqueous solutions incubated at low (5° C.) and elevatedtemperatures (40° C.) showed that less than 1.5% degradation occurs overa three month period as determined by HPLC analysis. The tetrasodiumsalt of P¹,P⁴-di(uridine 5′-) tetraphosphate was thus determined to havean excellent solubility and stability profile suitable forpharmaceutical applications.

Example 8 Toxicity of P¹,P⁴-di(uridine 5′-)tetraphosphate, TetrasodiumSalt in Animals

The nonclinical toxicologic profile of P¹,P⁴-di(uridine5′-)tetraphosphate, tetrasodium salt has been evaluated in a battery ofgenetic toxicology assays that include the bacterial reverse mutationassay, the in vitro mammalian cytogenetic test, the in vitro mammaliancell gene mutation test, and the micronucleus cytogenetic assay in mice.A study in rabbits examined local ocular tolerance and subchronic oculartoxicity after multiple daily administrations over a six-week period. Inaddition, P¹,P⁴-di(uridine 5′-)tetraphosphate, tetrasodium salt has alsobeen tested in two single-dose acute inhalation toxicity studies in ratand dog, and one single-dose acute intravenous toxicity study in dogs.

The results of these studies show that P¹,P⁴-di(uridine5′-)tetraphosphate, tetrasodium salt is nongenotoxic in a battery ofgenetic toxicology assays. No adverse findings were seen in the oculartoxicology studies. A low degree of acute toxicity was seen in singledose inhalation (rats, dogs) and intravenous (dogs) toxicity studies.P¹,P⁴-di(uridine 5′-)tetraphosphate, tetrasodium salt was thereforedetermined to have an excellent toxicology profile with a wide safetymargin for dosing in humans.

Example 9 Safety and Efficacy of P¹,P⁴-di(uridine 5′-)tetraphosphate,Tetrasodium Salt in Normal Human Volunteers

P¹,P⁴-di(uridine 5′-)tetraphosphate, tetrasodium salt was evaluated in aPhase I, double-blind, placebo-controlled, escalating dose, safety andtolerability study in 75 normal healthy male volunteers. Fortynon-smokers and 35 smokers were evaluated in 5 dosing cohorts of 16volunteers, comprised of 12 receiving a single aerosolized dose ofP¹,P⁴-di(uridine 5′-)tetraphosphate, tetrasodium salt (20-400 mg) and 4receiving placebo (normal saline). No serious adverse events werereported. There were no significant changes in FEV₁, FVC, MMEF, clinicallaboratory, 12-lead ECG, or urinalysis results in either the placebo oractive drug groups. In smokers, P¹,P⁴-di(uridine 5′-)tetraphosphate,tetrasodium salt produced a 2-fold to 7-fold dose-dependent increase inthe weight of sputum expectorated within 5 minutes of dosing, andstimulation of sputum expectoration was sustained over the next hour ofsputum collection. The effect of P¹,P⁴-di(uridine 5′-) tetraphosphate,tetrasodium salt to induce the expectoration of sputum in non-smokerswas also observed. In conclusion, P¹,P⁴-di(uridine 5′-)tetraphosphate,tetrasodium salt is safe and well-tolerated in normal male subjects andis effective in stimulating the expectoration of sputum when compared toplacebo.

Example 10 Method for the Production of Diuridine Tetraphosphate,Potassium Salt using Uridine 5′-Diphosphate

Uridine 5′-diphosphate disodium salt is dissolved in deionized water.The solution is passed through a Dowex 50Wx4 H⁺ (Dow Chemical) column.The fractions containing uridine 5′-diphosphate are pooled andneutralized with tributylamine (Aldrich, St. Louis, Mo.). Theneutralized fractions are concentrated to an oil form by using a rotaryevaporator at a bath temperature of 55-60° C. The oil is dissolved indry dimethylformamide (Aldrich) and then dried by concentrating to anoil using a rotary evaporator (55-60° C. bath temperature). This step isrepeated. The oil is dissolved in dimethylformamide and1,1-carbonyldiimidazole (Aldrich) is added. The solution is heated at50° C. An additional amount of activating agent is added and heatingcontinued. The solution is again concentrated to an oil form on a rotaryevaporator (bath temperature at 55-60° C.). The resulting oil isdissolved in deionized water. The solution is then loaded into a columnof Sephadex DEAE-A25 (Pharmacia, Upsala, Sweden; pre-swollen in 1.0 MNaHCO₃ and washed with 2 column volumes of deionized H₂O). The column iseluted with the following solutions in the following order: 60 L of 0.25M NH₄HCO₃, 120 L of 0.275M NH₄HCO₃, 40 L of 030 M NH₄HCO₃ and 40 L of0.35 M NH₄HCO₃. The fractions having sufficient amounts of purediuridine tetraphosphate were pooled as determined by HPLC analysis andconcentrated on a rotary evaporator (bath temperature at 55-60° C.). Theresulting residue is dissolved in deionized water (1.5 L) andconcentrated on a rotary evaporator. This step is repeated until excessof the bicarbonate buffer is removed. The resulting oil is dissolved ina sufficient amount of deionized water to form a ca. 10% solution, thesolution charged to a Dowex 50Wx4 H⁺ (Dow) column, which is prewashedwith potassium bicarbonate, and eluted with deionized water. Thefractions containing U₂P₄ are pooled and concentrated, then lyophilizedto yield U₂P₄ tetrapotassium salt as a solid.

Example 11 Method for the Production of Diuridine Tetraphosphate,Lithium Salt using Uridine 5′-Diphosphate

Uridine 5′-diphosphate disodiuim salt is dissolved in deionized water.The solution is passed through a Dowex 50Wx4 H⁺ (Dow Chemical) column.The fractions containing uridine 5′-diphosphate are pooled andneutralized with tributylamine (Aldrich, St. Louis, Mo.). Theneutralized fractions are concentrated to an oil form by using a rotaryevaporator at a bath temperature of 55-60° C. The oil is dissolved indry dimethylformamide (Aldrich) and then dried by concentrating to anoil form using a rotary evaporator (55-60° C. bath temperature). Thisstep is repeated. The oil is dissolved in dimethylformamide and1,1-carbonyldiimidazole (Aldrich) is added. The solution is heated at50° C. An additional amount of activating agent is added and heatingcontinued. The solution is again concentrated to an oil on a rotaryevaporator (bath temperature at 55-60° C.). The resulting oil isdissolved in deionized water. The solution is then loaded into a columnof Sephadex DEAE-A25 (Pharmacia, Upsala, Sweden; pre-swollen in 1.0 MNaHCO₃ and washed with 2 column volumes of deionized H₂O). The column iseluted with the following solutions in the following order: 60 L of 0.25M NH₄HCO₃, 120 L of 0.275M NH₄HCO₃, 40 L of 030 M NH₄HCO₃ and 40 L of0.35 M NH₄HCO₃. The fractions having sufficient amounts of purediuridine tetraphosphate were pooled as determined by HPLC analysis andconcentrated on a rotary evaporator (bath temperature at 55-60° C.). Theresulting residue is dissolved in deionized water (1.5 L) andconcentrated on a rotary evaporator. This step is repeated until excessof the bicarbonate buffer is removed. The resulting oil is dissolved ina sufficient amount of deionized water to form a ca. 10% solution, thesolution charged to a Dowex 50Wx4 H⁺ (Dow) column, which is prewashedwith lithium carbonate, and eluted with deionized water. The fractionscontaining U₂P₄ are pooled and concentrated, then lyophilized to yieldU₂P₄ tetralithium salt as a solid.

Example 12 Solubility and Stability of P¹,P⁴-di(uridine5′-)tetraphosphate, Tetralithium Salt

The solubility of P¹,P⁴-di(uridine 5′-)tetraphosphate, tetralithium saltin water is determined by adding portions of solid to a known volume ofdeionized water until the solution becomes turbid. Stability studies areperformed by incubating the solid or aqueous solutions at low (5° C.)and elevated temperatures (40° C.) for a period of time. Thetetralithium salt of P¹,P⁴-di(uridine 5′-)tetraphosphate is determinedto have an excellent solubility and stability profile suitable forpharmaceutical applications.

Example 13 Solubility and Stability of P¹,P⁴-di(uridine5′-)tetraphosphate, Tetrapotassium Salt

The solubility of P¹,P⁴-di(uridine 5′-)tetraphosphate, tetrapotassiumsalt in water is determined by adding portions of solid to a knownvolume of deionized water until the solution became turbid. Stabilitystudies are performed by incubating the solid or aqueous solutions atlow (5° C.) and elevated temperatures (40° C.) for a period of time. Thetetrapotassium salt of P¹,P⁴-di(uridine 5′-)tetraphosphate is determinedto have an excellent solubility and stability profile suitable forpharmaceutical applications.

Example 14 Toxicity of P¹,P⁴-di(uridine 5′-)tetraphosphate, TetralithiumSalt in Animals

The nonclinical toxicologic profile of P¹,P⁴-di(uridine5′-)tetraphosphate, tetralithium salt is evaluated in a battery ofgenetic toxicology assays that include the bacterial reverse mutationassay, the in vitro mammalian cytogenetic test, the in vitro mammaliancell gene mutation test, and the micronucleus cytogenetic assay in mice.A study in rabbits examines local ocular tolerance and subchronic oculartoxicity after multiple daily administrations over a six-week period. Noadverse findings are seen in the ocular toxicology studies. TheP¹,P⁴-di(uridine 5′-)tetraphosphate, tetralithium salt has an excellenttoxicology profile with a wide safety margin.

Example 15 Toxicity of P¹,P⁴-di(uridine 5′-)tetraphosphate,Tetrapotassium Salt in Animals

The nonclinical toxicologic profile of P¹,P⁴-di(uridine5′-)tetraphosphate, tetrapotassium salt is evaluated in a battery ofgenetic toxicology assays that include the bacterial reverse mutationassay, the in vitro mammalian cytogenetic test, the in vitro mammaliancell gene mutation test, and the micronucleus cytogenetic assay in mice.A study in rabbits examines local ocular tolerance and subchronic oculartoxicity after multiple daily administrations over a six-week period. Noadverse findings are seen in the ocular toxicology studies. TheP¹,P⁴-di(uridine 5′-)tetraphosphate, tetrapotassium salt has anexcellent toxicology profile with a wide safety margin.

Example 16 Method for the Production of Diuridine Tetraphosphate,Tetraammonium Salt using Uridine 5′-Diphosphate

The solution of tetraammonium salt of U₂P₄ in the ammonium bicarbonatebuffer was prepared and concentrated on a rotary evaporator to removethe excess of bicarbonate buffer according to Example 1.

The resulting oil is dissolved in a sufficient amount of deionized waterto form a ca. 10% solution, the solution is frozen and lyophilized togive P¹,P⁴-di(uridine 5′-) tetraphosphate, tetraamonium salt as a gummypowder in approximately 25% yield. The ¹H, ³¹P and ¹³C NMR data are inaccord with that of the structure of U₂P₄.

Example 17 Method for the Large Scale Production of DiuridineTetraphosphate, Disodiumdipotassium Salt from Diuridine Tetraphosphate,Tetraammonium Salt

Approximately one kilogram of P¹,P⁴-di(uridine 5′-)tetraphosphate,tetraammonium salt is dissolved in a sufficient amount of deionizedwater to form a ca. 10% solution. The amount of U₂P₄ present in thesolution is quantitated by comparison of the ultraviolet absorption atλ_(max) 263 nm of an accurately diluted sample with that of a standardsolution of P¹,P⁴-di(uridine 5′-)tetraphosphate at 0.1 mg/mL. Thissolution of P¹,P⁴-di(uridine 5′-) tetraphosphate, tetraammonium salt ofknown concentration is then cooled to 0° C. and treated with an ice coldsolution containing two equivalents of sodium acetate and twoequivalents of potassium bicarbonate in deionized water over two hours.The resulting solution is placed on a large rotary evaporator inportions and is concentrated under vacuum at below 60° C. to give athick liquid, which is dissolved in deionized water to give a ca. 10%solution. The evaporation and dilution processes are repeated two moretimes to remove volatile ammonium salts and then the residue isdissolved in water to form a ca. 10% solution which is lyophilized toyield U₂P₄, disodiumdipotassium salt as a white solid in excellent yieldand purity.

Example 18 Aqueous Stability and Solubility of P¹,P⁴-di(uridine5′-)tetraphosphate, Disodiumdipotassium Salt

The solubility of P¹,P⁴-di(uridine 5′-)tetraphosphate,disodiumdipotassium salt in water is determined by adding portions ofsolid to a known volume of deionized water until the solution becomesturbid. Stability studies are performed by incubating the solid oraqueous solutions at low (5° C.) and elevated temperatures (40° C.) fora period of time. The disodiumdipotassium salt of P¹,P⁴-di(uridine5′-)tetraphosphate is determined to have an excellent solubility andstability profile suitable for pharmaceutical applications.

Example 19 Toxicity of P¹,P⁴-di(uridine 5′-)tetraphosphate,Disodiumdipotassium Salt in Animals

The nonclinical toxicologic profile of P¹,P⁴-di(uridine5′-)tetraphosphate, disodiumdipotassium salt is evaluated in a batteryof genetic toxicology assays that include the bacterial reverse mutationassay, the in vitro mammalian cytogenetic test, the in vitro mammaliancell gene mutation test, and the micronucleus cytogenetic assay in mice.A study in rabbits examines local ocular tolerance and subchronic oculartoxicity after multiple daily administrations over a six-week period. Noadverse findings are seen in the ocular toxicology studies. TheP¹,P⁴-di(uridine 5′-)tetraphosphate, disodiumdipotassium salt has anexcellent toxicology profile with a wide safety margin.

Example 20 Method for the Production of Diuridine Tetraphosphate,Monopotassiumtrilithium Salt using Uridine 5′-Diphosphate

Uridine 5′-diphosphate disodium salt is dissolved in deionized water.The solution is passed through a Dowex 50Wx4 H⁺ (Dow Chemical) column.The fractions containing uridine 5′-diphosphate are pooled andneutralized with tributylamine (Aldrich, St. Louis, Mo.). Theneutralized fractions are concentrated to an oil form by using a rotaryevaporator at a bath temperature of 55-60° C. The oil is dissolved indry dimethylformamide (Aldrich) and then dried by concentrating to anoil using a rotary evaporator (55-60° C. bath temperature). This step isrepeated. The oil is dissolved in dimethylformamide and1,1-carbonyldiimidazole (Aldrich) is added. The solution is heated at50° C. An additional amount of activating agent is added and heatingcontinued. The solution is again concentrated to an oil form on a rotaryevaporator (bath temperature at 55-60° C.). The resulting oil isdissolved in deionized water. The solution is then loaded into a columnof Sephadex DEAE-A25 (Pharmacia, Upsala, Sweden; pre-swollen in 1.0 MNaHCO₃ and washed with 2 column volumes of deionized H₂O). The column iseluted with the following solutions in the following order: 60 L of 0.25M NH₄HCO₃, 120 L of 0.275M NH₄HCO₃, 40 L of 030 M NH₄HCO₃ and 40 L of0.35 M NH₄HCO₃. The fractions having sufficient amounts of purediuridine tetraphosphate are pooled as determined by HPLC analysis andconcentrated on a rotary evaporator (bath temperature at 55-60° C.). Theresulting residue is dissolved in deionized water (1.5 L) andconcentrated on a rotary evaporator. This step is repeated until theexcess of bicarbonate buffer is removed. The resulting oil is dissolvedin a sufficient amount of deionized water to form a ca. 10% solution,and the amount of U₂P₄ present in the solution is quantitated bycomparison of its ultraviolet absorption at λ_(max) 263 nm with that ofa standard solution of P¹,P⁴-di(uridine 5′-)tetraphosphate as in Example7. This solution of P¹,P⁴-di(uridine 5′-)tetraphosphate, tetraammoniumsalt of known concentration is then cooled to 0° C. and treated with anice cold solution containing three equivalents of lithium acetate andone equivalent of potassium bicarbonate. The resulting solution isplaced on a rotary evaporator and is concentrated under vacuum at below60° C. to give a thick liquid, which is dissolved in deionized water.The evaporation and dilution processes are repeated two more times toremove volatile ammonium salts and then the residue is dissolved inwater to form a ca. 10% solution which is lyophilized to yield U₂P₄,monopotassiumtrilithium salt as a white solid.

Example 21 Method for the Production of Diuridine Tetraphosphate,Monolithiummonopotassiumdisodium Salt using Uridine 5′-Diphosphate

Uridine 5′-diphosphate disodiuim salt is dissolved in deionized water.The solution is passed through a Dowex 50Wx4 H⁺ (Dow Chemical) column.The fractions containing uridine 5′-diphosphate are pooled andneutralized with tributylamine (Aldrich, St. Louis, Mo.). Theneutralized fractions are concentrated to an oil form by using a rotaryevaporator at a bath temperature of 55-60° C. The oil is dissolved indry dimethylformamide (Aldrich) and then dried by concentrating to anoil form using a rotary evaporator (55-60° C. bath temperature). Thisstep is repeated. The oil is dissolved in dimethylformamide and1,1-carbonyldiimidazole (Aldrich) is added. The solution is heated at50° C. An additional amount of activating agent is added and heatingcontinued. The solution is again concentrated to an oil on a rotaryevaporator (bath temperature at 55-60° C.). The resulting oil isdissolved in deionized water. The solution is then loaded into a columnof Sephadex DEAE-A25 (Pharmacia, Upsala, Sweden; pre-swollen in 1.0 MNaHCO₃ and washed with 2 column volumes of deionized H₂O). The column iseluted with the following solutions in the following order: 60 L of 0.25M NH₄HCO₃, 120 L of 0.275M NH₄HCO₃, 40 L of 030 M NH₄HCO₃ and 40 L of0.35 M NH₄HCO₃. The fractions having sufficient amounts of purediuridine tetraphosphate were pooled as determined by HPLC analysis andconcentrated on a rotary evaporator (bath temperature at 55-60° C.). Theresulting residue is dissolved in deionized water (1.5 L) andconcentrated on a rotary evaporator. This step is repeated until excessof the bicarbonate buffer is removed. The resulting oil is dissolved ina sufficient amount of deionized water to form an approximately 10%solution, and the amount of U₂P₄ present in the solution is quantitatedby comparison of its ultraviolet absorption at λ_(max) 263 nm with thatof a standard solution of P¹,P⁴-di(uridine 5′-)tetraphosphate as inExample 7. This solution of P¹,P⁴-di(uridine 5′-)tetraphosphate,tetraammonium salt of known concentration is then cooled to 0° C. andtreated with an ice cold solution containing one equivalent of lithiumformate, one equivalent of potassium bicarbonate, and two equivalents ofsodium ion in the form of sodium carbonate. The resulting solution isplaced on a rotary evaporator and is concentrated under vacuum at below60° C. to give a thick liquid, which is dissolved in deionized water.The evaporation and dilution processes are repeated two more times toremove volatile ammonium salts and then the residue is dissolved inwater to form an approximately 10% solution which is lyophilized toyield U₂P₄, monolithiummonopotassiumdisodium salt as a white solid.

Example 22 Solubility and Stability of P¹,P⁴-di(uridine5′-)tetraphosphate, Monolithiummonopotassiumdisodium Salt

The solubility of P¹,P⁴-di(uridine 5′-)tetraphosphate,monolithiummonopotassiumdisodium salt in water is determined by addingportions of solid to a known volume of deionized water until thesolution becomes turbid. Stability studies are performed by incubatingthe solid or aqueous solutions at low (5° C.) and elevated temperatures(40° C.) for a period of time. The monolithiummonopotassiumdisodium saltof P¹,P⁴-di(uridine 5′-)tetraphosphate is determined to have anexcellent solubility and stability profile suitable for pharmaceuticalapplications.

Example 23 Solubility and Stability of P¹,P⁴-di(uridine5′-)tetraphosphate, Monopotassiumtrilithium Salt

The solubility of P¹,P⁴-di(uridine 5′-)tetraphosphate,monopotassiumtrilithium salt in water is determined by adding portionsof solid to a known volume of deionized water until the solution becameturbid. Stability studies are performed by incubating the solid oraqueous solutions at low (5° C.) and elevated temperatures (40° C.) fora period of time. The monopotassiumtrilithium salt of P¹,P⁴-di(uridine5′-)tetraphosphate is determined to have an excellent solubility andstability profile suitable for pharmaceutical applications.

Example 24 Toxicity of P¹,P⁴-di(uridine 5′-)tetraphosphate,Monopotassiumtrilithium Salt in Animals

The nonclinical toxicologic profile of P¹,P⁴-di(uridine5′-)tetraphosphate, monopotassiumtrilithium salt is evaluated in abattery of genetic toxicology assays that include the bacterial reversemutation assay, the in vitro mammalian cytogenetic test, the in vitromammalian cell gene mutation test, and the micronucleus cytogeneticassay in mice. A study in rabbits examines local ocular tolerance andsubchronic ocular toxicity after multiple daily administrations over asix-week period. No adverse findings are seen in the ocular toxicologystudies. The P¹,P⁴-di(uridine 5′-)tetraphosphate,monopotassiumtrilithium salt has an excellent toxicology profile with awide safety margin.

The invention, and the manner and process of making and using it, arenow described in such full, clear, concise and exact terms as to enableany person skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the scope of the present invention as setforth in the claims. To particularly point out and distinctly claim thesubject matter regarded as invention, the following claims conclude thisspecification.

1. A pharmaceutical formulation comprising P¹,P⁴-di(uridine5′-)tetraphosphate, tetrasodium salt; in a pharmaceutically acceptablecarrier.
 2. The pharmaceutical formulation according to claim 1, whichis in the form of an aqueous, a gel, a patch, a cream, an ointment, asuppositiory, or a solid formulation.
 3. The pharmaceutical formulationaccording to claim 2, wherein the pharmaceutical formulation is in theform of an aqueous solution and comprises physiologically safeexcipients formulated to osmolarity between 250-350 mOsm and pH 5-9. 4.The pharmaceutical formulation according to claim 1, wherein thepharmaceutical formulation is sterile.
 5. A method of treating dry eyein a mammal by administering to the mammal the pharmaceuticalformulation according to claim
 1. 6. A method of treating retinaldetachment in a mammal by administering to the mammal the pharmaceuticalformulation according to claim
 1. 7. A pharmaceutical formulationcomprising P¹,P⁴-di(uridine 5′-)tetraphosphate, tetralithium salt; orP¹,P⁴-di(uridine 5′-)tetraphosphate, tetrapotassium salt; in apharmaceutically acceptable carrier.
 8. The pharmaceutical formulationaccording to claim 7, which is in the form of an aqueous, a gel, agel-like, a patch, a cream, an ointment, a suppositiory, or a solidformulation.
 9. The pharmaceutical formulation according to claim 7,wherein the pharmaceutical formulation is in the form of an aqueoussolution and comprises physiologically safe excipients formulated toosmolarity between 250-350 mOsm and pH 5-9.
 10. The pharmaceuticalformulation according to claim 7, wherein the pharmaceutical formulationis sterile.
 11. A method of treating dry eye in a mammal byadministering to the mammal the pharmaceutical formulation according toclaim
 7. 12. A method of treating retinal detachment in a mammal byadministering to the mammal the pharmaceutical formulation according toclaim
 7. 13. A method of treating dry eye in a mammal by administeringto the mammal an effective dry eye treatment amount of a compound ofFormula I:

each X is independently Li, Na, or K, provided that not all X's are Li,Na, or K.
 14. The method according to claim 13, wherein said compound isP¹,P⁴-di(uridine 5′-) tetraphosphate, tetra-alkali metal salt, in whichthe tetra-alkali metal cations of said salt are selected from the groupconsisting of: monosodium/tripotassium, disodium/dipotassium,trisodium/monopotassium, monopotassium/trilithium,dipotassium/dilithium, tripotassium/monolithium, monosodium/trilithium,disodium/dilithium, trisodium/monolithium,monosodium/monolithium/dipotassium, monosodium/dilithium/monopotassiumand disodium/monolithium/monopotassium.
 15. A method of treating retinaldetachment in a mammal by administering an effective dry eye treatmentamount of a compound of Formula I:

each X is independently Li, Na, or K, provided that not all X's are Li,Na, or K.
 16. The method according to claim 15, wherein said compound isP¹,P⁴-di(uridine 5′-) tetraphosphate, tetra-alkali metal salt, in whichthe tetra-alkali metal cations of said salt are selected from the groupconsisting of: monosodium/tripotassium, disodium/dipotassium,trisodium/monopotassium, monopotassium/trilithium,dipotassium/dilithium, tripotassium/monolithium, monosodium/trilithium,disodium/dilithium, trisodium/monolithium,monosodium/monolithium/dipotassium, monosodium/dilithium/monopotassiumand disodium/monolithium/monopotassium.